Inflammation is the body’s response to clear out pathogens upon infection or initiate tissue repair upon injury. It is a strictly regulated process that follows three general stages: initiation, execution and resolution. While essential to homeostasis, inflammation must be strictly regulated to avoid excessive tissue damage. My work focuses on a fairly recently appreciated family of molecules involved in resolution of inflammation. Siglecs (sialic acid binding immunoglobulin-like LECtins) are cell surface proteins, members of the immunoglobulin-like gene superfamily that bind sialic acid-containing glycans (sialoglycans). Selective expression of Siglecs on subsets of inflammatory cells and their potential to mediate inhibitory signaling through their ITIM motifs makes them appealing therapeutic targets to suppress ongoing inflammation and limit inflammatory tissue damage. Among the inhibitory siglecs is Siglec-8. Siglec-8 is selectively expressed on allergic inflammatory cells (eosinophils and mast cells) in the periphery and microglia in the brain. Crosslinking Siglec-8 by antibody or glycan ligands induces eosinophil apoptosis, inhibits release of inflammatory mediators by mast cells, and is proposed to inhibit microglial phagocytosis. Productive Siglec signaling requires the Siglec and its sialoglycan target (ligand) in tissues. My research spearheaded the discovery of endogenous Siglec-8 sialoglycan ligands on human airways and brain. Since Siglec-8 is uniquely human, I purified Siglec-8 ligands from human trachea, nasal lavage from patients with inflamed and non-inflamed airways, and brain cortex from non-demented and Alzheimer’s disease donors. I discovered that Siglec-8 ligands in all tissues are sialylated keratan sulfates with a characteristic glycan structure having a sialic acid adjacent to a sulfated galactose. This structure is appended to different proteins depending on the tissue. In each case, Siglec-8 ligand expression is upregulated under inflammatory conditions, suggesting tissue-level regulation of ongoing inflammation. Further studies will determine the role of regulated Siglec sialoglycan ligand expression in mediating microglial activity in dementia and allergic inflammatory cells in eosinophilic diseases. This work was done in the lab of Ronald L. Schnaar, Ph.D., in the Department of Pharmacology and Molecular Sciences, where we seek to further understand the role of glycans and glycan-binding proteins in diseases affecting humans.

I worked in Carol Greider’s laboratory, where we study how telomere length is regulated. Telomere length equilibrium contributes to fundamental cellular processes as well as cellular aging and cancer. Specifically, my thesis work focused on the telomere binding protein Rif1. Rif1 is a conserved protein known to regulate telomere length, origin firing and DNA repair, but the connection of these functions remained unclear. Through this work, we learned that Rif1 has at least two independent functions; we showed that it regulates telomere length through a separate mechanism than that of origin firing. We extended this discovery by mapping the region of Rif1 that is critical for its telomere regulation. This region of the protein has also been shown to be important for DNA repair. This result provides new insight into how Rif1 may regulate both telomere length and DNA repair. These findings expand on our fundamental understanding of proteins involved in telomere length regulation and our understanding of the coordination of telomere length regulation with other cellular processes.

Trigeminal neuralgia is a chronic, debilitating facial pain characterized by sudden, short and intense episodes of shooting, stabbing or shocklike pain in the face. The pain can be triggered by activities of everyday life, such as eating, drinking, talking or brushing teeth. Trigeminal neuralgia is so debilitating it was historically dubbed the “suicide disease” because patients would sometimes take their own life to end their suffering. Unfortunately, medical treatments for trigeminal neuralgia often fall short, and the only FDA-approved drug for trigeminal neuralgia carries a significant side effect profile. This collaborative study between the laboratories of Solomon Snyder and Michael Lim sought to understand the mechanisms underlying trigeminal neuralgia. Our discoveries provide insight into what causes pain in trigeminal neuralgia, and, importantly, identifies promising therapeutic targets to benefit patients suffering from this debilitating pain.

My research focuses on the persistence of HIV infection and its interplay with our immune system. The development of antiretroviral drugs changed the face of the HIV pandemic, transforming a devastating disease into a manageable chronic condition. However, despite successful treatment, HIV persists, integrated into the genome of infected cells as part of what we call the latent reservoir. Because of this reservoir, therapy must be continued indefinitely, requiring public health systems to deliver medications to all 38 million people living with HIV for life. Thus, understanding the mechanisms of HIV reservoir maintenance is paramount for the development of novel curative strategies. Previous studies showed that the proliferation of infected CD4+ T cells is a major cause of HIV persistence. In the laboratory of Robert and Janet Siliciano, I tried to untangle which forces drive HIV-infected clones to expand over time and survive. My thesis work demonstrated that immune responses to chronic antigens, such as those from other common viral infections, play a major role in determining the fate of infected cells. In other words, the T cells’ “day job” drives reservoir persistence. In most cases, the HIV provirus is just a passenger. Our work shows that HIV leaves a deep footprint on the immune system, which imposes huge challenges for future therapeutic interventions.

I did my research in the lab of Shigeki Watanabe. I study synaptic transmission: how neurons in the brain transfer information between each other by releasing neurotransmitter. Our approach to studying this in the Watanabe lab is unique: We stimulate neurons then freeze them at a specific moment in time so we can examine the structure of the synapse at that exact moment using high-resolution electron microscopy. Using this approach, we discovered that, within 15 milliseconds of neurons being stimulated, synaptic vesicles “dock” themselves into position to be ready to release neurotransmitter. This is a means for synapses to maintain and adjust themselves that hadn’t been considered before, and how this happens and what it does have become a whole line of inquiry for many labs in the past few years, including ours. We think these tiny movements of vesicles help support information processing in the brain.

In the lab of Takanari Inoue, we use and develop new biological tools to study and manipulate live cells. Cell signaling is crucial for all the processes of life at long and short timescales. Growth and development can take years, whereas fight or flight responses take less than a second. Studying how cells signal at fast timescales of seconds to minutes requires novel synthetic biology tools. Previously, all chemical and optogenetic systems allowed the end user to rapidly bring together two proteins with high specificity. To expand what we could achieve, I developed a novel chemically inducible trimerization (CIT) system to bring together three proteins of interest. CIT allows us to rapidly perturb membrane contact sites between organelles, and interrogate tri-organellar interactions. I am now using CIT and other synthetic biology tools to address the role of plasma membrane organization by the protein Pacsin2 on mast cell activation.

Our multidisciplinary research team of surgeons and engineers use 3D printing to rapidly prototype and test medical devices, established and developed by Sung Hoon Kang with Justin Sacks and me. At the height of the COVID-19 pandemic, we investigated ventilator solutions, and rapidly prototyped ventilator multiplexors and other ventilator-associated parts. This team was led by Sung Hoon Kang, Jamie Guest, and Julie Caffrey. Our work demonstrates the potential of multidisciplinary translational engineering teams, collaborations between multiple institutions, and of adapting emerging technologies to innovate and improve patient care.

Acidic pH is crucial for the function of intracellular organelles in the secretory and endocytic pathways. Furthermore, it is one of the pathological hallmarks of many diseases, including cerebral and cardiac ischemia, cancer, infection and inflammation. However, the molecular mechanisms of acid sensing and regulation are not fully understood. Exposure of cells to acidic conditions activates a ubiquitous proton-activated Clˉ channel, whose molecular identity has been a long-standing mystery in the field. Through an unbiased RNAi screen, the Qiu lab recently identified a novel and evolutionarily conserved membrane protein, PAC (encoded by PACC1 gene), as the proton-activated chloride channel. The discovery of such a new ion channel represents a major breakthrough, making it possible to reveal its fundamental structural and functional properties. I joined the Qiu lab when it opened its door at Hopkins five years ago, and decided to focus on this exciting new Clˉ gatekeeper. Taking advantage of single-particle cryo-electron microscopy, we solved two distinct cryo-EM structures of human PAC at a high-pH resting closed state (pH 8.0) and a low-pH, proton-bound, nonconducting state (pH 4.0). I identified key residues critical for pH sensing mechanism, channel inactivation and anion selectivity. My work provides the first glimpse of the molecular assembly of PAC, and a basis for understanding the mechanism of proton-dependent activation. We also showed that PAC, initially identified as a plasma membrane protein, traffics to the endosomes. It encodes a bona fide acid-sensitive Clˉ leak channel in endosomes and regulates endosomal pH, Clˉ level, and transferrin-receptor-mediated endocytosis. My research has uncovered a mechanism of endosomal Clˉ permeability and revealed a previously unappreciated complexity in endosomal biology.

The eukaryotic DNA is beautifully organized into a nucleosome comprising 147 bp DNA wrapped around an octamer of core histone proteins H2A, H2B, H3 and H4. The nucleosome is the basic repeating unit of a chromatin. The chromatin is decorated with a wide range of reversible histone post-translational modifications that regulate all processes that require access to DNA. Monoubiquitination of histone H2B (H2B-Ub) plays a role in transcription and DNA replication, and is required for the function of a protein complex that is responsible for the assembly and disassembly of nucleosomes called FACT. Dysregulation of H2B-Ub or FACT is associated with a variety of cancers. In Cynthia Wolberger’s lab, my study focused on understanding how FACT and the deubiquitinating enzyme Ubp10 work in concert to govern histone H2B deubiquitination. My work demonstrated that Ubp10 preferentially cleaves free-standing H2A/H2B-Ub dimers much faster than intact ubiquitinated nucleosomes, but that the addition of FACT stimulates Ubp10 activity on nucleosomes. Importantly, my work also demonstrated that disrupting the functions of these proteins in cells leads to defects in transcription and DNA replication. To get a better understanding of the reason behind Ubp10’s low activity on the nucleosomes, I solved the cryogenic electron microscopy (cryoEM) structures of Ubp10 bound to a ubiquitinated nucleosome. The structures revealed that Ubp10 makes several contacts with histones, ubiquitin, and severely alters the nucleosomal DNA at the nucleosome entry/exit site. Ubp10 docks onto the nucleosome in many conformations suggesting that the enzyme doesn’t bind nucleosomes in the correct register that promotes H2B deubiquitination without the help of FACT. The findings from my work highlight novel relationships between H2B monoubiquitination and the role of FACT in destabilizing the nucleosome to assist Ubp10 in H2B deubiquitination.

The work describes how early pancreatic precursor lesions (intraductal papillary mucinous neoplasms, or IPMNs) evolve, gather more and more mutations, and become pancreatic cancers. We demonstrated that certain lesions, often found in association with pancreatic cancers, are indeed the precursor to invasive disease. We also showed that mutations in genes associated with the TGF-beta pathway like SMAD4 and TGFBR2 were often mutated at the moment the precursor lesions transformed into cancer. This observation may impact how patients with IPMNs are treated. It is easy to detect these lesions with medical imaging. However, not everybody with such a precursor will develop cancer. Finding this mutation in i.e. DNA extracted from the fluid from these cysts can help decide whether these patients need surgery to remove the cyst. We also showed it takes around three years to develop cancer once you have a high-grade dysplastic precursor lesion: a window of opportunity to identify patients at risk for getting pancreatic cancer and treat them. I started the work in Laura Wood’s lab, where I did the wet lab work. However, I finished the analysis part in Victor Velculescu’s lab, where there was a lot of expertise on sequencing, analyzing and reconstructing phylogeny from very small neoplastic lesions.

My research focuses on the role of an atypical G protein-coupled receptor (GPCR) called Gpr116. In the Pluznick lab, we are focused on how Gpr116 and other GPCRs affect kidney physiology. Recently, I discovered that Gpr116 is a significant regulator of acid excretion by the kidney. More specifically, Gpr116 acts to inhibit runaway acid secretion in A-type intercalated cells in the collecting ducts. This discovery addresses a major gap in my field’s understanding of how A-type cells regulate acid secretion. Furthermore, since Gpr116 is an atypical GPCR with some unique structural features, we can begin to form hypotheses about the biomechanical cues that may reveal how Gpr116, and other similar GPCRs, affect our physiology.

I am a student in the Johns Hopkins Post-baccalaureate Research Education Program (PREP), working in Sandra Gabelli’s lab in the biophysics and biophysical chemistry department. My work is focused on tailoring g bisphosphonate inhibitors of farnesyl diphosphate synthase (FPPS) as lead compounds for new drugs that target leishmaniasis. I have determined the crystal structure of Leishmania major FPPS in complex with a series of nitrogen-containing bisphosphonates. We have measured their binding kinetics and performed cell assays to discern the features that are ideal for inhibition in vitro and in cells. This is important as, currently, there are no vaccines or drugs to prevent infection, and current treatments have high toxicity and generate drug resistance. Bisphosphonates represent a safer, compelling alternative for the treatment for leishmaniasis as they are currently being used for treatment of osteoporosis. We also hope to utilize these inhibitors against other parasitic diseases such as Chagas disease.

Gene therapy represents the potential to cure many monogenic diseases, including those of the liver. Adeno-associated virus (AAV) vectors have been used exclusively toward gene therapy of the liver in recent clinical trials, but AAV vectors present numerous disadvantages. Because of antibody responses toward the virus capsid, AAV vectors can’t be redosed. Furthermore, high doses of AAV can yield acute liver injury, and T-cell responses against the capsid can later eliminate gene-modified cells. Viral vectors also have enormous costs, limiting the application of gene therapy to more common diseases. To solve these issues, I worked with endoscopist Vivek Kumbhari to improve on a method of delivering naked plasmid DNA directly into the liver through the biliary system. Plasmid DNA could enter directly into hepatocytes through pores in the cell membrane when applied at high fluid pressures. Testing was performed in a human-sized pig model with clinical equipment in order to ensure translatability into patient testing. Crucially, I found that the percentage of liver cells expressing the delivered gene exceeded 20%, comparable to the best reported data of AAV in nonhuman primates. Pigs displayed no signs of liver injury, and were able to express transgene for several weeks post-injection. We believe this approach could pave the way for nonviral gene therapy to treat a variety of liver conditions.

CRISPR-Cas9 has catalyzed a biotechnological revolution through convenient and programmable genome editing. Cas9 itself, however, only performs the first step — site-directed induction of DNA damage. Completion of editing relies on the cell’s DNA repair machinery, which has been challenging to characterize due to the lack of control over DNA damage induction. In the laboratory of Taekjip Ha, we sought to tackle this problem by developing two systems for very fast light-mediated control over Cas9 activation and deactivation. First, we demonstrated Cas9 activation within seconds of light exposure, which allowed greatly improved kinetic measurements of DNA damage induction and repair. We discovered that Cas9-induced DNA damage could be detected within two minutes and repaired within 15 minutes, which is much faster than previously believed. Second, we demonstrated Cas9 deactivation within seconds of light exposure. Using this system, we discovered that only a few hours of CRISPR activity were sufficient for efficient genome editing. This system also greatly reduced editing at unintended “off-target” sites, which enhances the safety of genome editing. Together, my work in Dr. Ha’s lab opens the door for control of CRISPR-Cas9 as an effective method for studying genome editing and DNA repair.

The ability to adapt to a changing environment is constrained across development, with children and adolescents being generally more adaptable compared with adults and the elderly. This observation is captured by the concept of a “critical period.” Cognitive neuroscientists have long speculated on the existence of a critical period for social behavior in humans. My studies conducted in the Dölen Lab are the first to identify and characterize such a critical period. We demonstrate that: 1) animals are maximally sensitive to social reward learning cues during adolescence, 2) this sensitivity declines in adulthood, and 3) these adaptations correspond to a maturational downregulation of oxytocin-mediated synaptic plasticity in the nucleus accumbens, a key brain region of the reward system. Another important discovery is that the atypical psychedelic drug MDMA is able to reopen the social reward learning critical period in adulthood. Clinically, MDMA has been successfully used in treating people with post-traumatic stress disorder, and our studies provide a possible mechanism for its remarkable therapeutic properties. Opening the critical window for social reward behavior has significant implications for understanding the pathogenesis of neurodevelopmental diseases characterized by social impairments, as well as disorders that respond to social influence or are the result of social injury.

While working in Christopher Potter’s lab in the Department of Neuroscience, I discovered a novel regulatory mechanism that controls the expression of chemosensory genes, “odorant receptors,” on the mosquito’s nose. Odorant receptor regulation is an unexplored but important topic in vector biology because the precise control of this gene family influences a mosquito’s ability to locate and bite humans.

Through the use of big data, informatics and machine learning, we created tools with high predictive ability for identifying patients hospitalized with COVID-19 who are at high risk of progression to severe disease or death. First, we developed an interactive web tool (Severe COVID-19 Adaptive Risk Predictor) that provides risk predictions as well as explanations of the prediction logic in terms of interpretable decision trees. Afterward, we integrated our risk calculator into Epic at Johns Hopkins in record time to facilitate use within the clinical workflow and incorporation of risk scores as part of the electronic medical record. This work is important both in the context of the response to the COVID-19 pandemic as well as the broader field of machine learning in health care. I conducted this research in the lab of Scott Zeger, as part of the Johns Hopkins Individualized Health Initiative (Hopkins inHealth).

Retinal degeneration is the key pathological feature of many blinding diseases such as macular degeneration, retinitis pigmentosa and glaucoma. While current therapies can slow the progression of vision loss, there is no effective treatment to restore lost retinal neurons. One of the most promising potential therapies is to reprogram Müller glia that are already resident in the retina to replace retinal neurons lost to diseases. Müller glia from non-mammalian vertebrates, such as zebrafish, can be reprogrammed by injury into Müller glia-derived progenitor cells, which can regenerate diverse neuronal cell types, even into adulthood. In contrast, mammalian Müller glia, including humans, do not spontaneously regenerate neurons following injury. As a postdoctoral fellow in Seth Blackshaw’s lab, I performed comparative analysis of Müller glial response to injury across multiple species, zebrafish, chick and mice. We found that, in zebrafish and chick, genes selectively expressed in reactive Müller glia promoted the reprogramming of Müller glia into retinal neurons. In contrast, in mice, a dedicated gene regulatory network repressed the reprogramming of Müller glia. Disruption of nuclear factor I (NFI) transcription factors induces Müller glia to proliferate and generate neurons in adult mice following injury. These findings may aid in designing therapies to restore retinal neurons lost to degenerative diseases.

I have had the great opportunity to pursue my graduate research work in Andrew Holland’s lab. Our lab is fascinated with cell division and the molecular mechanisms that allow this process to happen precisely every time the cell divides. My research project focuses on cell divisions that occur during early brain development. Specifically, I was curious as to why mutations in proteins functioning at the centrosome — organelles that help form the bipolar spindle during mitosis, frequently lead to a brain developmental condition called microcephaly. Using mouse models carrying these mutations, I was able to show that during the pathogenesis of microcephaly, neural progenitor cells with centrosome defects take longer to complete mitosis, which in turn activate a signaling axis consisting of 53BP1, USP28 and TP53. Activation of this signaling pathway, collectively referred to as the mitotic surveillance pathway, leads to cell death in the developing brain, resulting in a smaller brain size with fewer neurons. Remarkably, removal of any components of the mitotic surveillance pathway is sufficient to restore neural progenitor proliferation and rescue brain size. These findings suggest that activation of the mitotic surveillance pathway is a central mechanism underlying microcephaly pathogenesis in human patients.

The hypothalamus is composed of a diverse array of neuronal and glial cell types, many of which are organized into spatially discrete clusters known as nuclei. The hypothalamus is essential for regulating a broad range of homeostatic physiological processes. Progress in this area has been hampered, however, by the fact that hypothalamic cell types thus far have remained quite poorly characterized. Still, less is known about how hypothalamic cell types acquire their identities during development. In the lab of Professor Seth Blackshaw, I have utilized rapidly advancing single-cell RNA-Sequencing (scRNA-Seq) technology to analyze the hypothalamus development at cellular resolution and profile changes in gene expression across all developmental stages. I integrate our findings of genes that control hypothalamic regionalization and neurogenesis and findings of gene regulatory networks that control cell identity to generate a Hypothalamic Developmental Database (HyDD). Our HyDD reference atlas is used to address various aspects of developmental biology: 1) comprehensive analysis of complex mutant phenotypes and 2) development of hypothalamic neuronal subpopulations.

In Beer lab, we study computational genomics and gene regulatory mechanisms. My research focuses on the regulation of higher order chromatin architecture in mammalian cells. I developed a computational model to predict the interaction of chromatins in three-dimensional space based on a process called loop extrusion. It explains how factors like CTCF and cohesin work together to form loops between distant regions quantitatively, which has important implications in nuclear DNA packing and gene expression regulation.

I am working on hair cell regeneration in murine cochlea. I discovered the mechanism of how supporting cells be reprogrammed and differentiated to hair cells. I also discovered some new progenitor genes in hair cell regeneration. This work was done in the lab of Angelika Doetzlhofer.

Maintaining genome integrity is an essential task of all cells, and imperfect repair of the damaged genome is responsible for a myriad of human diseases, ranging from aggressive cancers to defective immune systems. As a postdoc coadvised by Taekjip Ha and Bin Wu, I broadly explore the biophysical processes involved in DNA repair in living human cells. To achieve this goal, I developed a “very fast CRISPR” system that can induce double-strand DNA breaks (DSB) with unprecedented spatiotemporal control and specificity. vfCRISPR is a powerful enabling technique, akin to the “channelrhodopsins” in optogenetics, which permits ultrafast perturbation and subsequent interrogation of many physical and chemical processes during DSB repair. For example, this rapidly inducible CRISPR system can reveal the molecular interplay at the DSB junction, addressing how the kinetics of chromatin state switch, recruitment of repair factors, and noncoding RNA species determines DNA repair pathway choices and cell fate decision. Our studies provide valuable strategies and information complementary to traditional steady-state biochemical approaches.